POS0476 THE NUCLEAR RECEPTOR TR4 ORCHESTRATES CYTOSKELETAL ORGANIZATION IN A Gα12/ROCK-DEPENDENT MANNER TO PROMOTE MYOFIBROBLAST DIFFERENTIATION AND TISSUE FIBROSIS IN SYSTEMIC SCLEROSIS
Authors: L. Shen, Y. Zhang, Z. Honglin, J. H. W. Distler
Journal: Annals of the Rheumatic Diseases (2022)

Background Nuclear receptors (NR) are a family of transcription factors. Several members of the NR family are candidates for targeted intervention in inflammatory and fibrotic diseases (1-2). Testicular receptor 4 (TR4), also known as Nr2c2, has been shown to regulate fundamental cellular processes such as differentiation, proliferation and growth factor signaling (3-4). However, its role in fibrotic diseases has not been investigated so far. Objectives The aim of the present study was to characterize the role of TR4 in the pathogenesis of fibrotic tissue remodeling in SSc and to interrogate its underlying mechanism. Methods Expression of TR4 was quantified by RT-PCR, Western blot and immunofluorescence. The effects of TR4 knockdown on collagen production and myofibroblast differentiation were analyzed in cultured human fibroblasts and in three mouse models with fibroblast-specific knockout of TR4. RNA sequencing was performed in TGFβ-stimulated human dermal fibroblasts transfected with TR4 siRNA or non-targeting siRNA. The implication of TR4 in cytoskeleton regulation was analyzed by ROCK activity assays, stress fiber formation and quantification of the ratio of filamentous (F)-actin/ globular (G)-actin. The functional role of ROCK and Gα12 was analyzed using small molecule inhibitors and siRNA, respectively. Results TR4 expression was upregulated in fibroblasts in the skin of SSc patients and fibrotic mouse models. The expression of TR4 was upregulated in a TGFβ- and SMAD3-dependent manner. TR4 knockdown inhibited TGFβ-induced myofibroblast differentiation and collagen release, whereas overexpression of TR4 promoted fibroblast activation. Fibroblastspecific knockout of TR4 ameliorated experimental dermal fibrosis induced by bleomycin, adTBR and in sclGVHD with decreases in dermal thickening, myofibroblast counts and hydroxyproline content. The RNASeq of TR4 knockdown fibroblasts stimulated with TGFβ identified 651 differentially expressed genes as compared to control fibroblasts. Differentially expressed genes included central profibrotic genes such as ACTA2, COL3A1, CCL12. gProfiler enrichment analysis of the TR4-DEGs revealed enrichment of multiple GO, GSEA and Reactome terms related to ECM release, cytoskeleton organization and Rho GTPases activity. Indeed, knockdown of TR4 ameliorated the induction of ROCK activity by TGFβ stimulation and reduced the shift in the ratio from globular (G) actin to filamentous (F) actin. Knockdown of TR4 strongly reduced the expression of the G-protein alpha-12 (Gα12). ROCK inhibition by Y27632 or knockdown of Gα12 inhibited the induction of αSMA and stress fiber formation induced by overexpression of TR4. Conclusion TR4 is upregulated in SSc in a TGFβ-dependent manner to promote fibroblast activation. Inhibition of TR4 interferes with TGFβ-induced activation of ROCK, prevents cytoskeletal remodeling and fibroblast-to-myofibroblast transition and ameliorates experimental fibrosis. As nuclear receptors are common targets for therapeutic intervention, TR4 may offer potential for antifibrotic therapies. References [1]Avouac J, Palumbo-Zerr K, et al. The nuclear receptor constitutive androstane receptor/NR1I3 enhances the profibrotic effects of transforming growth factor beta and contributes to the development of experimental dermal fibrosis. Arthritis Rheumatol. 2014;66(11):3140-50. [2]Palumbo-Zerr K, Zerr P, et al. Orphan nuclear receptor NR4A1 regulates transforming growth factor-beta signaling and fibrosis. Nat Med. 2015;21(2):150-8. [3]Bookout AL, Jeong Y, Downes M, Yu RT, Evans RM, Mangelsdorf DJ. Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network. Cell. 2006;126(4):789-99. [4]Simandi Z, Cuaranta-Monroy I, Nagy L. Nuclear receptors as regulators of stem cell and cancer stem cell metabolism. Seminars in cell &amp; developmental biology. 2013;24(10-12):716-23. Disclosure of Interests Lichong Shen: None declared, Yun Zhang: None declared, ZHU Honglin: None declared, Jörg H.W. Distler Shareholder of: stock owner of 4D Science GmbH, Consultant of: JHWD has consultancy relationships and/or has received research funding from AbbVie, Actelion, BMS, Celgene, Bayer Pharma, Boehringer Ingelheim, JB Therapeutics, Sanofi-Aventis, Novartis, UCB, GSK, Array Biopharma and Active Biotech in the area of potential treatments of SSc.